The present disclosure relates to an improved fluorescent-quenching substrate comprising a phase inversion support associated with a plurality of opaque solids that are substantially non-reactive chemically with the phase inversion support and that are of a size sufficient to be partially or completely contained within, or irreversibly bound to, the phase inversion support. Such substrate may advantageously be employed in bioaffinity assays, including immunoassays and nucleic acid binding assays, which utilize luminescent tags, such as fluorescent tags. Such substrate further has use as filtration media to efficiently remove organic and/or inorganic material from fluids.
A great variety of assay systems have been developed to detect the presence and concentration of analytes in samples. For example, bioaffinity and enzymatically-activated catalysis reactions are widely used in medicine and science to analyze biological samples to detect and quantitize biological materials of concern. Many of these assay systems depend upon the binding of one chemical entity with the material of concern (or a modified form thereof) and detection of the conjugate, e.g., antigen-antibody, nucleic acid strand to complementary nucleic acid strand (“hybridization”), and protein-ligand conjugates. The conjugate is typically detected by way of a label providing a detectable signal which is attached to one or more of the binding materials. The conjugate is frequently quantitated by first determining the amount of label in the free and bound fractions, and then calculating the amount present using an algorithm and a set of standards to which the samples are compared.
The most common labels used in analyte binding assays are radioisotopes and luminescent compounds. Radioisotopes (Isotopic labeling) proffers considerably better detection in certain analyte systems than luminescent labeling. For example, the most sensitive methods for detecting nucleic acids typically involve the use of isotopic labeling, often involving radiolabelling with 32P. Luminescence is induced by energy transfer and refers to light emission that cannot be attributed merely to the temperature of the emitting body. Luminescent labels can be made to luminesce through photochemical (so-called, “photoluminescence”), chemical (so-called, “chemiluminescence”) and electrochemical (so-called, “electrochemiluminescence”) means. Photoluminescence, which includes fluorescence and phosphoresence, is a process whereby a material is induced to luminesce when it absorbs electromagnetic radiation such as visible, infrared or ultraviolet radiation. Chemiluminescence refers to luminescence occurring as a result of a chemical reaction without an apparent change in temperature. Electrochemiluminescence refers to luminescence occurring as a result of electrochemical processes.
In localizing particular sequences within genomic deoxyribonucleic acid (“DNA”), a transfer technique described by Southern is typically employed. DNA is digested, often using one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a gel. Conventionally the DNA is then denatured in situ and transferred from the gel to a solid support, the relative positions of the DNA fragments being preserved during and after the transfer to the solid support. The DNA attached to the solid support is then hybridized to radiolabelled DNA or ribonucleic acid (“RNA”), and autoradiography is used to locate the positions of bands complementary to the probe.
For many years, immobilization and hybridization of denatured DNA was carried out almost exclusively using nitrocellulose as a solid support. As time progressed, however, it became apparent that nitrocellulose was a less than an ideal solid-phase hybridization matrix, as nucleic acids are attached to the nitrocellulose support by hydrophobic, rather than by covalent interactions, and the nucleic acids are released slowly from the matrix during hybridization and washing at high temperatures. To overcome this problem, charge-modified cellulose supports, including DBM (diazobenzyloxymethyl)-cellulose and APT-cellulose, were introduced in the early 1980's to provide improved nucleic acid binding. These matrices, however, like nitrocellulose itself, also suffer from a significant disadvantage in that they become brittle when dry and cannot survive more than one or two cycles of hybridization and washing, i.e., “reprobing.”
Extensive use today is made of polyamide matrices, in particular nylon matrices, as solid support for immobilization and hybridization of nucleic acids. Various types of nylon are known to bind nucleic acids irreversibly and are far more durable than nitrocellulose. As nucleic acids can be immobilized on nylon in buffers of low ionic strength, transfer of nucleic acids from gels to a nylon matrix can be carried out electrophoretically, which may be performed if transfer of DNA by capillary action or vacuum is inefficient. Two basic types of nylon membranes are commercially available, unmodified nylon and charge-modified nylon. Charge-modified nylon is preferred for transfer and hybridization as its increased positively-charged surface has a greater capacity for binding nucleic acids (See, e.g., U.S. Pat. No. 4,473,474, the disclosure of which is herein incorporated by reference). Nylon membranes must be treated to immobilize the DNA after it has been transferred, as by way of thorough-drying, or exposure to low amounts of ultraviolet irradiation (254 nm).
While polyamide matrices have found considerable use in isotopic assay systems, such matrices have not found widespread use in fluorescent assay systems. This is likely due to the fact that fluorescent assay systems employing polyamide substrates demonstrate less than desirable sensitivity. Such reduction in sensitivity has been attributed primarily to two factors—background fluorescence produced by the nylon itself, and light scattering by solid materials in contact with the reaction media (such as substrates to which reactants are attached, or walls of the containers in which measurements are made). Polyamides, such as nylon, show light-stimulated endogenous fluorescent emissions and light reflection which can coincide with the range of UV-visible wavelengths emitted from fluorophore-tagged analytes. When light in the excitation waveband causes fluorescence of the support material, interference with detection occurs if the emission waveband of the fluorophore overlaps the same.
While isotopic assays on the whole are very sensitive, they suffer from a number of disadvantages. Primarily, use of any radioisotope automatically invokes health concerns and a host of regulatory duties with respect to waste disposal, safety, handling, reporting and licensing. While present luminescent assays proffer an alternative to isotopic labeling, the sensitivity of such assays is still not within a range desired by many in the biomedical, genetic research and drug discovery communities. Additionally, isotopic labeling cannot be used in multiplex assays, in which two or more nucleic acid probes which have been separately labeled each with their own unique colored luminescent label can be simultaneously hybridized, then simultaneously detected on an array of bound nucleic acid targets affixed to the polymeric substrate. Multiplexing saves significant cost and time when compared to the traditional steps of stripping and reprobing when performing multiple queries on a given array of targets. Multiplexing also reduces error and signal degradation that is associated with multiple reprobings.
U.S. Pat. Nos. 4,837,162 and 4,921,878 to Rothman et al., disclose a dye-modified polyamide material for use in luminescent assays which is said to both reduce the background fluorescence due to the polyamide, as well as light scattering by solid materials in contact with the reaction media. By reducing such properties, it asserted that such polyamide substrates allow improved detection of fluorescent emissions from fluorophore-tagged analytes as compared to untreated polyamide substrates. These patents disclose dyeing the polyamide material with a reactive dye, that is, a dye that contains a functional group that chemically reacts with the material being dyed (See, Column 6, Lines 54-57, of U.S. Pat. No. 4,921,878), having an absorbance spectrum selected to overlap the excitation and/or emission waveband of light generated by the polyamide substrate. It is asserted that acid-reactive dyes capable of integrally binding to the polyamide do not adversely affect the properties of the polyamide substrate. Acid-reactive dyes of the azo class are said to be particularly useful and to be readily manipulated to absorb light in the necessary waveband. Non-metallic acid reactive dyes are said to be preferred.
While the dyed-polyamides disclosed in U.S. Pat. Nos. 4,837,162 and 4,921,878 have been known for over a decade, such polyamides have not found widespread acceptance in luminescent assay systems. The failure of such substrates to dominate the market may relate to the less than desirable fluoresecence quenching that has been able to be produced following the disclosures of U.S. Pat. No. 4,837,162. It may also relate to the difficulty in identifying dyes that are both chemically-reactive with respect to the nylon and chemically non-reactive with typical analytes of interest. It is also possible that the dyes specified in these references interfere with binding of the biomolecules with the native nylon surface, either by creating an unfavorable surface for adsorption of the biomolecule, or directly competing with nylon for adsorption. One possible outcome of this competition is the creation of a biomolecule:azo dye complex which is less stable and more easily extracted and lost from the nylon surface, thus detrimentally affecting the analysis.
The biomedical and scientific communities would eagerly use fluorescent assays, as opposed to isotopic assays, if the detection sensitivity of fluorescent assays can be enhanced without increasing the potential for undesired chemical reactions. While sensitivity can be increased if the substrate on which fluorescent assays are performed does not fluoresce upon such exposure, isolation of such substrates having widespread usefulness (with respect to numerous analytes) has so far eluded the art. There is a need, therefore, for improved substrates for use in luminescent assays which lead to greater sensitivity for detecting analytes in a sample.